ABSTRACT Human papillomavirus (HPV) infections are highly prevalent in men and women. High-risk (HR) HPV types 16 & 18 (HPV16/18) are responsible for ~90% of all anal cancers, whose incidence has risen by 96% in the last 2- 3 decades primarily because of the HIV/AIDS epidemic. Indeed, HIV-infected men who have sex with men (HIV+MSM) are the population at highest risk of anal cancer with an incidence rate of 137 per 100,000 compared with 2 per 100,000 among HIV-uninfected men. Current anal cancer screening begins with an anal Papanicolaou (Pap) test to look for abnormal cytology, and in some clinics include HR-HPV DNA testing, which detects infection. If cytologic abnormalities of atypical squamous cells of unknown significance or higher are found on the anal Pap, patients are referred for high-resolution anoscopy (HRA), an invasive procedure to visualize and remove abnormal tissue for histology. Unfortunately, anal Pap screening under-estimates the grade of dysplasia compared to histology. Furthermore, HR-HPV DNA screening has limited utility among the HIV+MSM population because prevalence of anal HPV infection is so high (~80%). Screening individuals for HR-HPV DNA, although sensitive for detecting infection, is not specific for predicting risk of anal dysplasia. A more specific companion diagnostic is urgently needed to help determine when someone should be referred for HRA or not. To that end, we will be the first to assess the following combined measures of HPV gene expression from anal specimens: a) methylation status of all 15 CpG sites within the long control region (LCR) upstream of E6/E7 oncogenes, b) intra-epithelial levels of HR-HPV E6/E7 mRNA, and c) HPV 16/18 E6 protein expression. We hypothesize that combining 1 or more of these biomarkers with the anal Pap test, rather than the HR-HPV DNA test, would improve specificity for predicting AIN2+. To evaluate this, we will conduct a cross-sectional study of a diverse population of HIV+MSM with abnormal anal Pap results, who are undergoing HRA at clinics here in the District of Columbia, the city with the highest HIV prevalence rate in the U.S. Prior to the HRA procedure, 3 anal swabs will be collected: DNA will be extracted from one and used to assess CpG methylation patterns, an epigenetic biomarker, in HPV16/18 LCR, while the 2nd will be placed into liquid-based cytology media for HPV DNA genotyping, HR-HPV E6/E7 mRNA expression, and HPV16/18 E6 oncoprotein detection, and the 3rd will be analyzed directly for HPV16/18 E6 oncoproteins. Results of these tests will be compared independently for their ability to predict AIN2+ from HRA guided biopsies. HPV DNA genotyping data will permit us to characterize distribution of anogenital HPV types found in HIV+MSM here in DC. Identifying a more specific biomarker will improve the accuracy of predicting anal dysplasia in this high-risk group by providing stronger evidence to justify HRA if positive, or reduce the rate of unnecessary HRA if negative. Discovering a better companion diagnostics remains important in the HPV vaccine era because many HIV+MSM were never vaccinated and vaccine uptake continues to be lower than the target goals.